The cell envelope of Gram-negative bacteria contains two membranes, inner (IM) and outer (OM), and an aqueous compartment termed the periplasm that is located between them. A long-term goal of my lab has always been to understand the mechanisms of envelope biogenesis using Escherichia coli as a model system. This proposal concerns OM biogenesis and the stress responses that maintain cell envelope physiology. All of the components of the OM, phospholipids (PL), lipopolysaccharide (LPS), lipoproteins, and ?-barrel proteins (OMPs), are synthesized in the cytoplasm or the inner leaflet of the IM. We have identified the essential proteins required to transport LPS and OMPs across the periplasm and assemble these molecules in the OM. Recent work has provided functional insights into the periplasmic chaperones and the OM components of these two assembly machines, LptDE and BamABCDE. In addition, we have demonstrated that the Bam complex can export portions of the lipoprotein RcsF onto the cell surface by forming a highly interlocked complex in such a way that a short, unstructured, charged transmembrane domain of the lipoprotein is threaded through the lumen of an OMP ?-barrel where it is protected from the hydrophobic membrane interior. To study OMP assembly we will test our hypothesis that the parvulin domains of the major periplasmic chaperone function in a regulatory manner. To probe Bam complex function we will identify the signals in unfolded OMPs that the Bam complex recognizes and we will find mutant substrates that will slow or stall the folding process so that intermediates can be characterized. Mutations that specifically affect formation of lipoprotein/OMP complexes will be identified and characterized. Our studies on LPS assembly will continue with a focus of how LPS molecules exit the LptD-E complex in the OM. Mutations that hinder LPS movement will be identified and these will be characterized using photocrosslinking methods that track LPS movement. With regard to envelope stress, we will test our hypothesis that the RcsF/OMP complex senses LPS structural defects directly, again using photocrosslinking probes to monitor RcsF movement within the OMP lumen. We will also continue our studies with the Mla system that removes PLs from the outer leaflet of the OM and probe the mechanisms that maintain the OM barrier when cells are starved for nutrients. The essential Bam and Lpt proteins represent attractive new drug targets. Indeed, these targets are accessible at the cell surface and thus not protected either by the OM barrier or by efflux pumps. But what is particularly intriguing is that even if they did not kill, Bam or Lpt inhibitors would disrupt the OM barrier rendering strains more sensitive to existing antibiotics and thus could be especially effective in combination therapies. The more we learn about the OM barrier and how it is made, the more rational and sophisticated our approaches to find small molecule inhibitors.